Oral ingestion of food leads to the secretion of insulin and insulin counter regulatory hormones in a concerted effort to control blood glucose levels by increasing glucose and free fatty acid uptake by the liver, muscle and adipose tissue, and to reduce gluconeogenesis from the liver.
Insulin secretion is modulated by secretagogue hormones, termed as incretins, which are produced by enteroendocrine cells. Glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) account for almost all of the incretin effect. GIP, but not GLP-1, is ineffective in diabetic subjects. There is thus a great deal of interest in using GLP-1 and its analogues in therapeutic treatments for diabetes [for detailed discussion of GLP-1 physiology, see reviews Kieffer and Habener (1999); Doyle and Egan (2001); Hoist (1999); Perfetti and Merkel (2000); Nauck (1997); Gutniak (1997); Drucker (2001).
A summary of the current knowledge of GLP-1 physiology is provided below. Extensive information and the references to specific aspects are provided in the reviews cited above. GLP-1 is a 30 aa peptide derived from proglucagon, a 160 aa prohormone. Actions of different prohormone convertases in the pancreas and intestine result in the production of glucagons and other ill-defined peptides, whereas cleavage of proglucagon results in the production of GLP-1 and GLP-2 as well as two other peptides. The aa sequence of GLP-1 is 100% homologous in all mammals studied so far, implying a critical physiological role. GLP-1 (7–37) OH is C-terminally truncated and amidated to form GLP-1 (7–36) NH2. The biological effects and metabolic turnover of the free acid GLP-1 (7–37) OH, and the amide, GLP-1 (7–36) NH2, are indistinguishable. By convention, the numbering of the amino acids is based on the processed GLP-1 (1–37) OH from proglucagon. The biologically active GLP-1 is the result of further processing: GLP-1 (7–36) NH2. Thus the first amino acid of GLP-1 (7–37) OH or GLP-1 (7–36)NH2 is His7.
In the gastrointestinal tract, GLP-1 is produced by L-cells of intestinal, colonic and rectal mucosa, in response to stimulation by intraluminal glucose. The plasma half-life of active GLP-1 is <5 minutes, and its metabolic clearance rate is around 12–13 minutes (Holst, 1994). The major protease involved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV (CD26) which cleaves the N-terminal His-Ala dipeptide, thus producing metabolites, GLP-1 (9–37) OH or GLP-1 (9–36) NH2 which are variously described as inactive, weak agonist or antagonists of GLP-1 receptor. GLP-1 receptor (GLP-1R) is a G protein coupled receptor of 463 aa and is localized in pancreatic beta cells, in the lungs and to a lesser extent in the brain, adipose tissue and kidneys. The stimulation of GLP-1R by GLP-1 (7–37) OH or GLP-1 (7–36)NH2 results in adenylate cyclase activation, cAMP synthesis, membrane depolarization, rise in intracellular calcium and increase in glucose-induced insulin secretion (Holz et al., 1995).
GLP-1 is the most potent insulin secretagogue that is secreted from the intestinal mucosa in response to food intake. Fasting levels of immunoreactive GLP-1 in humans is about 5–10 pmol/L and rises to 25 pmol/L post-prandially (Perfetti and Merkel, 2000 vide supra). The profound incretin effect of GLP-1 is underscored by the fact that GLP-1R knockout mice are glucose-intolerant (Scrocchi et al.,). The incretin response of iv infused GLP-1 is preserved in diabetic subjects, though the incretin response to oral glucose in these patients is compro mised. GLP-1 administration by infusion or sc injections controls fasting glucose levels in diabetic patients, and maintains the glucose threshold for insulin secretion (Gutniak et al. 1992; Nauck et al., 1986; Nauck et al., 1993). GLP-1 has shown tremendous potential as a therapeutic agent capable of augmenting insulin secretion in a physiological manner, while avoiding hypoglycemia associated with sulfonylurea drugs.
Other important effects of GLP-1 on glucose homeostasis are suppression of glucagon secretion and inhibition of gastric motility (Tolessa et al., 1998). GLP-1 inhibitory actions on alpha cells of the pancreas leads to decreases in hepatic glucose production via reduction in gluconeogenesis and glycogenolysis (D'Alessio et al., 1997). This antiglucagon effect of GLP-1 is preserved in diabetic patients.
The so-called ileal brake effect of GLP-1, in which gastric motility and gastric secretion are inhibited, is effected via vagal efferent receptors or by direct action on intestinal smooth muscle. Reduction of gastric acid secretion by GLP-1 contributes to a lag phase in nutrient availability, thus obviating the need for rapid insulin response. In summary, the gastrointestinal effects of GLP-1 contribute significantly to delayed glucose and fatty acid absorption and modulate insulin secretion and glucose homeostasis.
GLP-1 has also been shown to induce beta cell specific genes, such as GLUT-1 transporter, insulin receptor (via the interaction of PDX-1 with insulin promoter), and hexokinase-1. Thus GLP-1 could potentially reverse glucose intolerance normally associated with aging, as demonstrated by rodent experiments (Perfetti and Merkel. 2000. vide supra). In addition, GLP-1 may contribute to beta cell neogenesis and increase beta cell mass, in addition to restoring beta cell function (Wang et al., 1997; Xu et al., 1999).
Central effects of GLP-1 include increases in satiety coupled with decreases in food intake, effected via the action of hypothalamic GLP-1R. A 48 hour continuous sc infusion of GLP-1 in type II diabetic subjects, decreased hunger and food intake and increased satiety (Toft-Nielsen et al., 1999). These anorectic effects were absent in GLP-1R knock out mice (Scrocchi et al., 1996 vide supra).
In summary, the diverse roles played by GLP-1 in maintaining metabolic homeostasis, makes it an ideal drug candidate for treating diabetes, obesity and metabolic syndrome.
Stability of GLP-1 in Circulation
GLP-1 released from the L-cells of the intestine, in response to food, enters portal circulation. It is rapidly cleaved by DPP IV (CD26) to release GLP-1 (9–37) or GLP-1 (9–36) amide, both of which are less active at GLP-1R. According to some reports, they may act as antagonists of GLP-1R and GLP-1 effects on gastrointestinal motility. The half-life of circulating GLP-1 was found to be about 4 minutes (Kreymann et al., 1987). Dipeptidyl-peptidase IV (DPP IV, EC 3.4.14.5, CD26), designated CD26, is an extracellular membrane-bound enzyme, expressed on the surface of several cell types, in particular CD4+ T-cells, as well as on kidney, placenta, blood plasma, liver, and intestinal cells. On T-cells, DPP IV has been shown to be identical to the antigen CD26. CD26 is expressed on a fraction of resting T cells at low density, but is strongly up-regulated following T-cell activation (Gorrell et al., 2001). Recent results indicate that CD26 is a multifunctional molecule that may have an important functional role in T-cells and in overall immune system modulation. CD26 is associated with other receptors of immunological significance found on the cell surface, such as the protein tyrosine phosphatase CD45 and adenosine deaminase (ADA). DPP IV exerts a negative regulation of glucose disposal by degrading GLP-1 and GIP, thus lowering the incretin effect on beta cells of the pancreas.
DPP IV-resistant Analogues of GLP-1
DPP IV cleaves the Ala-Asp bond of the major circulating form of human GLP-1 (human GLP-1 (7–36) NH2: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln -Ala-Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2) (SEQ ID NO:1), releasing an N-terminal dipeptide.
Substitution of Ala with Gly (Deacon et al., 1998; Burcelin et al., 1999), Leu, D-Ala and other amino acids, was shown to protect GLP-1 from DPP IV degradation and potentiates its in-vitro and in-vivo insulinotropic actions (Xiao et al., 2001).
Deletion of the amino-terminal histidine, or of the NH2 group of His7, decreased receptor affinity and potency of the analogue (Adelhorst et al., 1994; Xiao et al. 2001 vide supra; Siegel et al., 1999).
U.S. Pat. No. 5,545,618 teaches that N-terminal modifications using alkyl and acyl modifications also produced DPP IV resistant analogues. More specifically, His7 substitution by N-alkylated (C1–6) or N-acylated (C1–6) L-/D-amino acids resulted in analogues possessing DPP IV-resistance. However, the examples given in this patent only cover acetyl and isopropyl groups.
Covalent coupling of unsaturated organic acids, such as trans-3-hexenoic acid, also produces DPP IV-resistant GLP-1 analogs that potently reduce hyperglycemia in oral glucose tolerance tests in mice (Xiao et al. 2001 vide supra). Furthermore, His7 can be replaced by α-substituted carboxylic acids, one of the substituents being a 5- or 6-membered ring structure (e.g. imidazole), in order to confer DPP IV resistance (WO 99/43707). Insertion of 6-aminohexanoic acid (AHA) after His7 was shown to confer DPP IV resistance, while retaining receptor affinity and insulinotropic efficacy in vivo (Doyle et al., 2001).
Numerous GLP-1 analogs demonstrating insulinotropic action are known in the art. These variants and analogs include, for example, GLP-1(7–36), Gln9-GLP-1(7–37), D-Gln9-GLP-1(7–37), acetyl-Lys9-GLP-1(7–37), Thr16-Lys18-GLP-1(7–37), and Lys18-GLP-1(7–37). Derivatives of GLP-1 include, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (WO 91/11457 (1991); EP 0 733,644 (1996); and U.S. Pat. No. 5,512,549 (1996)). It has also been demonstrated that the N-terminal histidine residue (His7) is very important to the insulinotropic activity of GLP-1 (Suzuki et al., 1988).
Modification of His7 by alkyl or acyl (C1–6) groups, and replacement of His with functionally-equivalent C5–6 ring structures appears to confer DPP IV resistance. However, current information does not divulge if all covalent modifications of His7 also retain GLP-1 function in vitro and in vivo.
There thus remains a need to develop modified GLP-1 peptides having increased biological potency.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.